Although biological functionalization and applications of quantum dots (QDs) have been described for previously, the focus has been on utilizing the QD photoluminescence as a fluorophore or fluorometric label or marker in a bio-assay. Earlier studies observed an increase in QD photoluminescence when proteins are specifically coordinated to the QD surface, see Mattoussi et al, Self-assembly of CdSe—ZnS quantum dot bioconjugates using an engineered recombinant protein, J Am. Soc. 122: 12142-12150 (2000); Medintz et al, Self-assembled nanoscale biosensors based on quantum dot FRET donors, Nat. Mat. 2: 630-638 (2003)). Medinz et al, coordinated several different proteins to the surface of QDs and monitored the effect on photoluminescence. The proteins were self-assembled or coordinated to the surface of the QDs using the HIS-Zn mechanism or metal affinity. The MBP lacking the N-terminal 5-HIS sequence was unable to coordinate to the QD and thus unable to increase QD photoluminescence. The other proteins were able to specifically coordinate to the QD surface and increase the QD photoluminescence with slightly different efficacies. Those proteins were believed to passivate QD surface charge.
Myoglobin, with a slightly smaller molecular weight, containing an internal Heme moiety (with possible electrochemical properties, as opposed to the apo-myoglobin lacking Heme) has more moderate, modulated, differing effects on the QD photoluminescence as opposed to the apo-myoglobin. This coordination of proteins increases the QDs photoluminescence in a concentration dependant manner. The coordination of proteins at or near the actual QD surface enhances the QD photoluminescence response to excitation energy by passivating the electric field effects brought by the set of charges deriving from the COOH groups arrayed around the nanoparticle surface. The surface charge of QDs (on the inorganic cores) is critical in determining whether they will be emissive or not. This has been attributed to Auger ionization process (Efros et al, Phys. Rev. Lett. 78, 1110 (1996).
Additional studies have focused on using QDs as the fluorescence resonance energy transfer (FRET) donor portion of a FRET pair consisting of QDs that are not necessarily conjugated to any other entity, coordinated to protein acceptor dyes. See Medintz et al., 2003: Clapp, et al. Fluorescence resonance energy transfer between quantum dot donors and dye labeled protein acceptors. J. Am. Chem. Soc. 126: 301-310, 2004. Although the references explore the uses of QDs in biological applications, there was no discussion of a biological strategy or mechanism that could be used for the specific control of QD properties as desired.
Additionally, although not biological in nature, the results of the Guyot-Sionnest group studies suggest that charge and charge injection can be used as a mechanism to control QD photoluminescence. Furthermore, the response of biological entities in a controlled manner has been harnessed and applied to development of sensors and other biological devices. This includes binding events, enzymatic events, hybridization events, cleavage events, as well as changes in conformation to name but a few.
The photoluminescence emission from individual CdSe QDs under continuous low intensity laser illumination is intermittent, with single dots exhibiting binary on-off emission patterns. See Nirmal, et al. Nature 383, 802 (1996), Banin, et al. J. Chem. Phys. 110, 1195 (1999) and Kuno, et al, Chem. Phys. 112, 3117 (2000). Random ionization followed by neutralization of the nanocrystals under sustained laser irradiation is the likely cause of this on-off effect. In this theory, ionized nanocrystals are dark while neutral QDs are emissive (Efros et al., 1996). According to this model, a charged quantum dot is dark as a result of an efficient Auger process. Instead of emitting a photon, an exciton in a charged dot efficiently transfers its energy to another charge in the dot, which is excited to a higher electronic state and then relaxes back to its ground state non-radiatively. Although the exact ionization and neutralization process is not yet understood, its effect is dramatic: A charged dot does not emit light. This binary turning on and off of the fluorescence by charging is a powerful property that can now be controlled in creating active nanocrystal based fluorescent nanosensors. A small QD capacitor (a thin layer of quantum dots sandwiched between two electrodes) was controllably charged by applying a voltage (Woo et al, Reversible charging of CdSe nanocrystals in a simple solid-state device, Adv. Mat. 14 (15): 1068, 2002). As the applied voltage reaches a critical point allowing charge injections QD bulk fluorescence becomes quenched by as much as 70%. Photodarkening of ensembles of CdSe—ZnS nanocrystals embedded in thin film structures, either close packed or dispersed in a matrix of ZnS, has also been observed. The darkening in these films was speculated to be the result of ionizing a substantial fraction of the dots in the films (J. Rodriguez-Viejo, et al. J. Appl. Phys. 87, 8526 (2000)).
P. Guyot-Sionnest et al. showed that by exposing a solution of colloidal CdSe QDs to strong redox molecules, an electron could be injected in the valence band of these nanocrystals. This electron (n-type) doping translated in a substantial loss (bleaching) of the first absorption peak accompanied by loss of the photoluminescence emission. These experiments were further extended to other systems and sample configurations. For example, they showed that applying a potential to a set of QDs dispersed in an electrolytic solution, results in bleaching of the first absorption peak and reduces the photoluminescence emission when the applied voltage exceeds a threshold value. Results were further extended to solid films of QDs.
Biological applications of luminescent colloidal semiconductor nanoparticles or quantum dots are only beginning to be realized. Bio-functionalization of QDs and their use as a fluorescent marker in a biological assay have been demonstrated. With the burgeoning field of nanotechnology growing every year and exploring the interface between biology and materials, many new uses and devices for QDs will be found. These new nanotechnological uses in research and other fields will require specific control of QD properties such as photoluminescence, energy absorbance and other specific QD effects/characteristics or phenomena. Currently there exists no specific and controllable manner to achieve this except for varying the amount of energy used to excite the QDs, the amount of charge used to inject electrons into the QD or through direct exposure to chemicals. Many of these methods exert gross effects on the QD for research or other purposes. Although bio-functionalization of QDs has been demonstrated, with the field growing annually, the focus has been on using the QDs as biolabeling fluorophores or energy donor in fluorescence energy transfer. None of the bio-functionalization reported has been intended for, created for or has realized control of specific QD properties.
There is a need in the art for a method for controlling the properties of luminescent colloidal quantum dots. This need is addressed by a method that is designed to be useful for controlling the properties of luminescent quantum dots through a change in state of a biological entity either in close proximity to or attached to the surface of a QD. The control of various QD properties of interest will be affected by these biological entities in a controlled manner. The control of QD properties can be affected by these biological entities in a gross or on/off absolute manner. The control of QD properties can be affected by these biological entities in a modulated or desired or controllable manner. The control of QD properties can be affected by these biological entities through the addition of an external chemical, agent or effect which in turn affects this biological entity and its subsequent control of QD properties. The control of QD properties can be affected by these biological entities in a concurrent or consecutive manner. Although control of QD properties can be affected by many different biological entities which change state in many different ways, the same basic principles and overall architecture and conceptual scheme apply. These control mechanisms may be utilized to control all aspects of QD properties of interest including, but not limited to QD absorption, emission, magnetic, charge, redox and other properties